ES3034860T3 - Novel peptides and their use in diagnosis - Google Patents

Abstract

New peptides have been developed for the diagnosis of CagA+ H. pylori infection or for predicting the risk of gastric cancer. These peptides bind to antibodies from CagA+ H. pylori patients with high specificity and sensitivity, and can be used, for example, in a diagnostic kit.

Description

DESCRIPTION

New peptides and their use in diagnosis

Field of the invention

The present invention relates to novel peptides from the CagA protein of Helicobacter pylori. These peptides can be used to improve the prevention, diagnosis, and treatment of bacterial infections, as well as for assessing the risk of gastric cancer.

Background

Helicobacter pylori is a bacterium commonly found in the stomach. Some H. pylori strains carry the CagA (cytotoxicity-associated antigen A) gene, which encodes virulence factors. The CagA gene encodes the CagA protein, a bacterial oncoprotein of 1,140 to 1,180 amino acids that translocates into stomach epithelial cells at the site of infection. After translocation, it affects intracellular signaling pathways in epithelial cells.

H. pylori bacteria carrying the CagA gene are associated with an increased risk of developing gastric cancer, and the presence of anti-CagA antibodies is associated with an increased risk of developing gastric cancer later in life. Early detection of CagA+ H. pylori infection may increase cancer survival, as eradicating the infection in infected individuals reduces the risk of gastric cancer. Therefore, using methods to identify individuals who are carriers of CagA+ H. pylori can diagnose individuals at high risk of gastric cancer and thus help prevent its development.

However, existing serological methods for CagA+ H. pylori infections are not clinically useful, mainly because of their lack of specificity. Samples with high levels of false positives indicating generalized reactivity to CagA antibodies exist, even in individuals not infected with H. pylori, or in individuals infected with H. pylori strains that do not express CagA. Thus, there is no clinically useful diagnostic test with sufficient specificity and sensitivity (Yamaoka et al., J Clin Microbiol 1998:36:3433; Yamaoka et al., Gastroenterology 1999:117:745; Figueiredo et al., J Clin Microbiol 2001:39:1339).

Therefore, diagnostic tests for H. pylori CagA+ with improved diagnostic properties, e.g., higher specificity and sensitivity, are needed.

Furthermore, there is great variability in DNA sequences between different H. pylori isolates. Certain CagA variants are more closely associated with gastric cancer risk. Therefore, it would also be useful to be able to identify the types of CagA strains.

There is also a need for CagA peptides that specifically bind to antibodies, particularly antibodies that bind to the CagA protein.

Document US2016/030510 discloses a 14-amino acid peptide derived from CagA and a 29-amino acid peptide derived from CagA. These peptides are used in the diagnosis and treatment of tauopathies.

Document CN104147589B discloses the 18-amino acid cecropin peptide AWRK6, which is used in the diagnosis and treatment of septic shock.

YASUDAET AL"A novel diagnostic monoclonal antibody specific for Helicobacter pylori CagA of East Asian type", APMIS, vol. 117, no. 117. 12, November 17, 2009 (2009-11-17), pages 893-899, XP055157605, discloses a highly specific ELISA assay utilizing a monoclonal antibody that specifically recognizes CagA-positive East Asian H. pylori, wherein the antibody is generated against an 18 aa peptide (AINRKIDRINKIASAGKG) specific for East Asian CagA. It is known that H. pylori producing the East Asian variant of the CagA protein is more virulent than that producing West Asian CagA.

Summary of the invention

This document provides information on CagA-derived peptides that are useful for diagnostic applications related to H. pylori-associated diseases, including the identification of individuals at high risk of developing gastric cancer. Individuals infected with H. pylori produce antibodies against H. pylori proteins, including CagA. Therefore, the presence of CagA-specific antibodies indicates H. pylori infection.

From all CagA peptides present in infected individuals, the inventors have defined 1) an immunogenic subset that induces an antibody response (see Table 1, where 34% of the protein length is immunogenic). Many peptides have also been found to react with serum from uninfected patients (white bars in Fig. 1). Within the subset of immunogenic peptides, the inventors have identified 2) a smaller subset of peptides with diagnostic potential, and finally, from this subset of diagnostic peptides, the inventors have identified 3) the crucial amino acid sequence(s) common to the most diagnostic peptides. In other words, diagnostic capability is not derived solely from the presence or absence of peptides in infected individuals, but also critically from a small subset of immunogenic peptides that consistently elicit an antibody response that is absent in uninfected individuals.

By using high-precision serology, with resolution at the peptide rather than protein level, the inventors have identified peptides that elicit a strong antibody response only in H. pylori CagA+ carriers, while excluding peptides that cause false positives due to a cross-reactive antibody response in individuals without H. pylori CagA+ infection. The diagnostic peptides identified by the inventors therefore have high sensitivity and specificity, as confirmed by the ROC curve AUC values, and will therefore be useful for diagnostic applications.

In a first aspect of the invention, which does not form part of the claimed invention, there are provided peptides comprising or consisting of amino acid sequences selected from the group consisting of SEQ ID NO 1 to SEQ ID NO 2. Preferably, said peptides consist of a maximum of 25 amino acids, more preferably of a maximum of 15 amino acids and even more preferably of a maximum of 10 amino acids. In a preferred embodiment, the peptides comprise or consist of sequences selected from the group consisting of SEQ ID NO 2-7, or even more preferably from the group consisting of SEQ ID NO 2-5.

These new peptides have the advantage that they can be used for diagnosis, and more specifically, for the diagnosis of CagA-positive H. pylori. Thus, diagnosis with these peptides results in few false positives.

The identified minimal binding region is also useful for detecting CagA-specific antibodies. Due to its short length, background binding will be low. Furthermore, these peptides are short, so they can be manufactured at low cost.

In a second aspect of the invention, a peptide is provided comprising amino acid sequences selected from the group consisting of SEQ ID NO 1 to SEQ ID NO 5, said peptide consisting of a maximum of 25 amino acids, for use in the diagnosis of Helicobacter pylori infection and prediction of the risk of gastric cancer.

In a third aspect of the invention, which does not form part of the claimed invention, there is provided a kit comprising a peptide according to the first aspect of the invention or a mixture of peptides according to the second aspect of the invention. The kit is preferably a diagnostic kit, more specifically for the diagnosis of CagA-positive H. pylori or the prediction of gastric cancer risk.

In a fourth aspect of the invention, there is provided a diagnostic method comprising the steps of a) providing a sample from a subject, the subject being a human; b) contacting said sample with a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO 1 to SEQ ID NO 5, said peptide consisting of a maximum of 25 amino acids; c) detecting the specific binding of antibodies in the sample to the peptide, wherein the method is not practiced in a human body.

In a fifth aspect of the invention, there is provided a method for detecting CagA-binding antibodies of H. pylori in a sample from a subject. The method comprises contacting a biological sample with a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO 1 to SEQ ID NO 5, said peptide consisting of a maximum of 25 amino acids, and detecting the binding of the antibodies in the sample to the peptide.

The sample may be blood, serum, plasma or tissue, for example, a gastric tissue sample.

Brief description of the drawings

Fig. 1. Identification of 18 different linear CagA epitopes on B cells by peptide microarray analysis. The score of each peptide (n = 1172 peptides) on the array is shown as a vertical bar at the start position of the CagA sequence (x-axis). Black bars correspond to results from serum of H. pylori-infected individuals, while white bars correspond to results from serum of non-H. pylori-infected individuals. Notably, many peptides also showed reactivity with serum from non-infected individuals (white bars).

Fig. 2. ROC curve AUC levels of all peptides analyzed from the 18 identified CagA epitopes (n = 1144 peptides). Fig. 2a: Results represented in a box plot including median, interquartile range, and outliers. Fig. 2b: Results for each individual peptide are shown, grouped by epitope. In both 2a and 2b, the AUC of an unhelpful diagnosis (AUC = 0.5) is indicated by a dashed horizontal line.

Fig. 3. ROC curve AUC scores for all peptides containing crucial sequence motifs. Data are represented as median, interquartile range, and outliers. When only one peptide has been analyzed, only the median (horizontal line) is represented. The sequence motif designations are identical to the sequence names in Table 4:

BT_300: IINQKVTDKVDNLNQ (SEQ ID NO 13) (at least 12 of the 15 amino acids are identical, n = 298 peptides); BT_301: EPIYA (SEQ ID N 0 8)(n = 270);

BT_302: EPIYAK (SEQ ID NO 9) (n = 16);

BT_303: EPIYAQ (SEQ ID NO 10) (n = 21);

BT_304: EPIYT (SEQ IDNO 11)(n= 21);

BT_305: EPIYAT (SEQ ID NO 12) (n = 196);

BT_306: FXLKRHX (SEQ ID NO 1) (n = 246); BT_307: FXLKKHX (SEQ ID NO 2) (n=34);

BT_308: FXLKQHX (SEQ ID NO 3) (n=1);

BT_309: YXLKRHX (SEQ ID NO 4) (n=3);

BT_310: IXLKRHX (SEQ ID NO 5) (n=1);

BT_311: FXLRRYX (SEQ ID NO 6) (n=1);

BT_312: FXLRRSX (SEQ ID NO 7) (n=7).

AUC = 0.5 is indicated by a dashed horizontal line.

Detailed description

Occasionally, reference is made herein to a range of sequences. This refers to all sequences within that range. Thus, for example, "SEQ ID NO 2 to SEQ ID NO 5" refers to SEQ ID NOs 2, 3, 4, and 5. The sequences are written using standard one-character notations for amino acid residues. The amino acid residues are preferably linked by peptide bonds.

Some peptides herein may exhibit sequence variability. Thus, certain sequences may specify a position in the sequence where any amino acid may be present. In the sequence listing, this may be indicated as X or Xaa. X or Xaa may be substituted with any amino acid, preferably any L-amino acid, including amino acids resulting from post-translational modifications, such as citrulline. The amino acids do not have to be naturally occurring amino acids. Since bulky side chains may impede antibody binding, it is preferable that the amino acids do not have bulky side chains. A suitable molecular weight of the amino acids may be between 85 D and 300 D, preferably between 89 D and 220 D.

In general, peptides may comprise or consist of an amino acid sequence selected from the group consisting of SEQ ID NO 1 to SEQ ID NO 5. In certain embodiments, amino acids may be substituted in a conserved manner, for example, by substituting hydrophobic amino acids with other different hydrophobic amino acids, or polar amino acids with other different polar amino acids.

In a preferred embodiment, a peptide is used that comprises or consists of one of SEQ ID NO 1 to SEQ ID NO 5, for example, SEQ ID NO 1, 2, 3, 4 or 5 (Table 4). These sequences comprise the minimal binding regions of certain antibodies. In a preferred embodiment, the peptide comprises or consists of an amino acid sequence selected from SEQ ID NO 1 to SEQ ID NO 5.

In an even more preferred embodiment, the peptide comprises or consists of a sequence selected from SEQ ID NO 1 to SEQ ID NO 5. These peptides have the advantage of greater diagnostic accuracy, since they induce a strong antibody response in a high percentage of individuals with H. pylori CagA+ infection. All of these peptides (SEQ ID NO 1 to SEQ ID NO 5) are related to the same epitopes (epitope 12 and 14), and around 95% of all H. pylori CagA+ isolates in the world carry at least one of these sequence variants. In addition, the peptides share the following structural characteristics:

• They all have seven amino acid residues.

• They all have a hydrophobic residue in the first position (F, Y or I).

• They all have an x in the second position.

• They all have an L in the third position.

• They all have a K or an R (positive side chains) in the fourth position.

• They all have an x in the seventh position.

Examples of useful peptides comprising the sequences SEQ ID NO 1 to SEQ ID NO 5 include, but are not limited to, the sequences SEQ ID NO 129 to SEQ ID NO 170, SEQ ID NO 186 to SEQ ID NO 187, and SEQ ID NO 266 to SEQ ID NO 279.

In an even more preferred embodiment, the peptide comprises or consists of a sequence selected from SEQ ID NO 1, 2, 3, 4 and 5. Examples of useful peptides comprising these sequences are described in Tables 2 and 3.

Preferably, the peptide is 25 amino acids or less in length, such as 20 or 15 amino acids. A shorter peptide may be desirable because it results in less non-specific binding (by antibodies) and therefore less background. However, in some cases, a longer peptide may be desirable to allow exposure of the epitope and thus allow unhindered antibody binding, or to allow folding of the peptide. Thus, preferably, the peptide has 14 amino acid residues, more preferably 13 amino acid residues, or even more preferably 12, 11, 10, 9, 8, 7, 6, or 5 amino acid residues (6 applies only to SEQ ID NOs 8, 11, 9, 10, and 12, and 5 applies only to SEQ ID NOs 8 and 11).

Preferably, the peptide binds specifically (in the immunological sense) and with high affinity to antibodies, preferably also to antibodies that bind to the H. pylori CagA protein. The interaction between an antibody and a peptide is said to exhibit "specific binding" or "preferential binding" in the immunological sense if it reacts or binds more frequently, rapidly, for a longer duration, and/or with affinity to specific cells or substances than to other cells or substances. When an antibody binds with greater affinity, avidity, ease, and/or duration than to other substances, the antibody is said to "specifically bind" or "preferentially bind" to the peptide. Binding can be determined by any suitable method. Binding can be determined by methods known in the art, for example, ELISA, surface plasmon resonance, Western blotting, or other methods described herein (see below). Such methods can be used to determine suitable amino acid lengths or sequences of the peptide.

Preferably, the peptide used has both high diagnostic specificity and high diagnostic sensitivity. In any diagnostic test, these two properties depend on the level used as the cutoff for positive tests. To assess diagnostic accuracy without relying on established cutoff values, the receiver operating characteristic (ROC) curve can be used. An ROC curve plots the true positive rate (sensitivity) against the false positive rate (1-specificity) as the cutoff varies from 0 to infinity. The overall diagnostic accuracy is then estimated using the area under the ROC curve (AUC-ROC). Preferably, the use of the peptide has an AUC-ROC of at least 0.55, for example, an AUC-ROC of at least 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 0.96, 0.97, 0.98, 0.99 or an AUC-ROC of 1.00. Preferably, the use of the peptide has an AUC-ROC of at least 0.85, and more preferably an AUC-ROC of 1.

As used herein, the term "peptide" is used to refer to peptides, proteins, protein fragments, and the like, including peptidomimetic compounds. The term "peptidomimetic" refers to a peptide-like molecule that possesses the activity of the peptide upon which it is structurally based, such activity being to bind specifically and with high affinity to antibodies that bind to the CagA protein. Such peptidomimetics include chemically modified peptides, peptide-like molecules containing unnatural amino acids (see, e.g., Goodman and Ro, Peptidomimetics for Drug Design, in "Burger's Medicinal Chemistry and Drug Discovery," vol. 1 (ed. M. E. Wolff; John Wiley & Sons, 1995), pp. 803-861). Various peptidomimetics are known in the art, including, for example, peptide-like molecules containing a restricted amino acid. In certain embodiments, cyclic peptides may be used.

Peptides can be isolated peptides, that is, peptides in a form other than that in which they naturally exist, for example, in a buffer, in a dry form awaiting reconstitution, as part of a kit, etc.

In some embodiments, the peptides are substantially purified, meaning that they contain substantially no other proteins, lipids, carbohydrates, nucleic acids, or other biological materials with which they are naturally associated. For example, a substantially pure peptide can be at least about 60% by dry weight, preferably at least about 70%, 80%, 90%, 95%, or 99% by dry weight.

The peptides of the present invention may be presented in salt form. Suitable acids and bases capable of forming salts with the peptides are well known to those skilled in the art and include inorganic and organic acids and bases.

The peptides can be presented in a solution, for example, an aqueous solution. Such a solution may comprise suitable buffers, salts, protease inhibitors, or other suitable components known in the art.

The peptides may be linked (e.g., coupled, fused, or linked, directly or indirectly) to one or more additional moieties known in the art. Non-limiting examples of such moieties include peptide or non-peptide molecules such as biotin, poly-His tags, GST, FLAG, linkers, or spacers. The linkage may be a covalent or non-covalent bond. The linkage may be, for example, through terminal cysteine residues or chemically reactive linker, the biotin-avidin system, or poly-His tags. For example, the peptide may be linked via a peptide bond to a single biotin-conjugated Usine residue, wherein the Usine is biotinylated through the epsilon-amino groups of its side chain, such as in the peptide example H-XXXXXXXXXXXXXXX(K(Biotin))-NH2, where X indicates the amino acids of the peptide.

The associated moiety can be used to bind or link the peptide, improve purification, enhance expression of the peptide in a host cell, facilitate detection, stabilize the peptide, etc. In the case of short peptides bound to a substrate, for example, a solid phase, it may be desirable to use a linker or spacer to ensure that the peptides are exposed to the antibodies so that they can bind.

Peptides can be bound to a substrate that binds them. The substrate can be, for example, a solid or semi-solid carrier, a solid phase, a support, or a surface. Peptides can be bound to a solid support. Examples include microtiter plates, such as 96-well plates, which include microspheres or plate wells, as well as the surfaces of laboratory-on-a-chip diagnostic devices. Binding can be covalent or non-covalent and can be facilitated by a peptide-associated moiety that allows covalent or non-covalent binding, such as a moiety with high affinity for components bound to the carrier, solid phase, support, or surface. For example, the biotin-avidin system can be used.

The peptides can be used to detect CagA-specific antibodies from H. pylori in samples obtained from subjects, the method comprising contacting a biological sample with a peptide, as described herein, and detecting binding of antibodies in the sample to the peptide. The peptide can be bound to a substrate that fixes it, as described herein, for example, attached to a solid support. The method can include incubation to allow binding, washing, and detection of antibodies, as described herein. Methods for detecting antibody binding are described below, and include, for example, ELISA.

Peptides can be used for diagnosis, particularly in the diagnosis of H. pylori infection and gastric cancer. H. pylori CagA infection is known to correlate with an increased risk of gastric cancer.

Thus, the peptides can be used to assess a subject's risk of developing gastric cancer. The risk of developing gastric cancer can include the risk of progressing from not having gastric cancer to having gastric cancer at any stage, of progressing from a benign disease state to a malignant state, or of progressing from a less malignant state to a more malignant state. Thus, the risk can include the risk of having gastric cancer or of developing gastric cancer in the future. In a preferred embodiment, the peptide is used to assess a subject's risk of developing gastric cancer in the future. The peptides can also be used for the diagnosis of other diseases associated with H. pylori infection, such as peptic ulcer disease, dyspepsia, and immune thrombocytopenic purpura (ITP).

The diagnosis can be made by any suitable method. In a preferred method, antibodies in a sample from the subject are caused to bind to the peptide, and such binding is detected. The subject can be a human or an animal, preferably a human. In vitro binding of the subject's antibodies to the peptide indicates that the subject's immune system has generated antibodies against that particular peptide and, therefore, that the peptide, and by extension, H. pylori CagA, is present in the subject.

The method may comprise the steps of (1) isolating a sample of body fluid or tissue likely to contain antibodies from a subject or providing such a sample in vitro; (2) contacting the sample with a peptide under conditions that allow efficient formation of a specific peptide-antibody complex (for specific binding of the peptide to the antibody), for example, by reacting the sample with the peptide or incubating it; and (3) analyzing the contacted (reacted) sample for the presence of an antibody-peptide reaction (for example, by determining the amount of antibody-peptide complexes). The method may include one or more washing steps, as is known in the art. Steps 2 and 3 are preferably performed in vitro, that is, by using the sample, once isolated from the subject, on a sample previously isolated from a subject.

The sample may be any suitable sample, for example, a sample of blood, serum, plasma, saliva, mucous secretion, ascitic fluid, or a sample of similar body fluids or tissues.

Antibody responses to peptides can be detected using various immunological/serological methods. Suitable formats for detecting the presence of antibodies using peptides include peptide microarrays, ELISA, chromatography, Western blot, lab-on-a-chip formats, single or multiplexed bead-based immunoassays, and so on.

Typically, these methods include a process of verifying peptide binding to the stationary phase (such as the well of an ELISA plate or the surface of a microsphere), adding the sample to be tested in the liquid phase, allowing the antibodies to bind, and then a washing process to remove unbound antibodies.

Antibody binding can be detected in vitro using labeled secondary antibodies that bind to a specific type of human antibody, e.g., IgG, IgA, IgG1, IgG2, or IgG3, IgG4. In ELISA, secondary antibodies are labeled with an enzyme, such as horseradish peroxidase (HRP) or alkaline phosphatase (AP). Secondary antibodies can be from a species other than humans, e.g., rabbit or goat.

Alternatively, fluorescent or radioactive markers can be used.

The person skilled in the art can easily optimize the protocol for the use of peptides in an ELISA test with regard to the secondary antibody used, its dilution, buffer, blocking solution, washing solution, etc. An example of an ELISA protocol using plates is presented below: polystyrene microtiter plates are coated with optimal concentrations, determined by checkerboard titration, of the peptides of interest dissolved in PBS at room temperature overnight. After two washes with PBS, the wells are blocked with 0.1% (w/v) bovine serum albumin-PBS at 37 °C for 30 min. Subsequent incubations are carried out at room temperature, washing the plates three times with PBS containing 0.05% Tween (PBS-Tween) between incubations. Serum or other body fluid samples are added in duplicate or triplicate at initial dilutions of, for example, 1/10, and diluted, for example, in a three-fold dilution series. Previously tested control samples that present antibodies against the peptides are used as positive controls. Samples with known antibody concentrations can be used to create a standard curve. Wells to which only PBS-Tween is added are used as negative controls to determine background values. After 90 minutes of incubation at room temperature, HRP-labeled rabbit anti-IgA or anti-human IgG antibodies are added and incubated for 60 minutes. The plates are then read in a spectrophotometer 20 minutes after the addition of H<2>O<2> and ortho-phenylenediamine dihydrochloride in 0.1 M sodium citrate buffer, pH 4.5. The endpoints for each sample titration are determined by reciprocal interpolation as the dilution whose absorbance at 450 nm is, for example, 0.4 times higher than the background. Alternatively, the absorbance can be used as the final reading. Those skilled in the art will understand that this ELISA protocol is merely an example and that many variations and modifications are possible.

Alternatively, in one embodiment, B cells are isolated from the subject and tested for their ability to produce antibodies that bind to the peptide. This can be done using ELISPOT, ALS (antibodies in lymphocyte secretions), or similar methods.

The diagnosis can also be made by detecting the presence of the CagA protein in a tissue sample from a patient using specific antibodies against a peptide selected from the peptides comprising or consisting of SEQ ID NOs 32-330, SEQ ID NOs 1-5, in particular SEQ ID NOs 2-5. The sample is preferably a gastric tissue sample. The person skilled in the art can produce antibodies with the desired binding specificity. The antibody can be polyclonal or monoclonal, with monoclonal antibodies being preferable. The antibody can be used in any format useful for detecting proteins, for example, Western blot, ELISA, immunohistochemistry, etc. These antibodies can be used for the diagnostic methods described herein.

The method can give two possible results: presence of H. pylori infection or absence of H. pylori infection. H. pylori infection can be determined, for example, based on the signal cutoff value in the assay. Intermediate results may also occur, i.e., uncertain results that require further investigation, resampling, or reanalysis of the samples.

Once the presence of a CagA+ H. pylori infection has been confirmed, it may be useful to treat the infection, for example, to reduce the risk of the subject developing gastric cancer. Treatment can be carried out by methods known in the art, for example, with the use of antibiotics. For various reasons, including the low availability of active antibiotics in the stomach and problems with antibiotic resistance, there are various antibiotic treatment regimens for H. pylori infection, and their effectiveness often varies by region of the world. Typically, treatment regimens include at least two different antibiotics selected from the groups of macrolides, beta-lactams, nitroimidazoles, tetracyclines, and fluoroquinolones, with or without the addition of bismuth subcitrate potassium, preferably one antibiotic from each group. One or more antibiotics may be administered in combination with a proton pump inhibitor. Treatment includes administration of the proton pump inhibitor omeprazole and the antibiotics amoxicillin and clarithromycin for 7 to 14 days.

Therefore, there is also provided a method for preventing gastric cancer comprising the steps of 1) diagnosing a subject as described herein and 2) treating the subject's H. pylori infection. Preferably, the treatment is performed such that the subject is free of H. pylori infection.

Once the presence of CagA+ H. pylori infection has been confirmed, further examination to assess for the presence of early or advanced gastric cancer may also be helpful. This may be relevant for all patients, but is especially important for patients known or suspected to be at high risk for gastric cancer, such as patients from countries with a high risk of gastric cancer, smokers, and/or individuals with a close family history of gastric cancer. This examination can be performed by gastroscopy, in which the stomach wall is examined to assess for the presence of gastric cancer. If a gastric tumor is detected, it can be treated by endoscopic resection if it is found in an early stage, or by surgery if it is found in an advanced stage.

Alternatively, the method can be used as a follow-up to routine gastroscopy. If endoscopy and/or subsequent histopathological examination reveal the presence of precancerous lesions in the stomach, for example, through an elevated OLGA score, the method can be used to inform the patient's further management. This may include a recommendation for follow-up gastroscopy at appropriate intervals. For example, if a CagA+ H. pylori infection has been confirmed, it may be beneficial to perform follow-up gastroscopy at shorter intervals than if no CagA+ H. pylori infection is present.

Peptides can be synthesized by methods known in the art. They can be obtained in pure form and in large quantities by organic synthesis, such as solid-phase synthesis. Methods for peptide synthesis are well known in the art, for example, using a peptide synthesis machine. Of course, peptides can be ordered from a company specializing in peptide synthesis.

Peptides can also be of animal, plant, bacterial, or viral origin. The peptide can then be purified from the organism, as is known in the art. The peptide can be produced by recombinant technology, for example, using eukaryotic cells, bacterial cells, or viral expression systems. For further details, see Current Protocols in Molecular Biology (Ausubet et al., Eds.), John Wiley & Sons, NY (current edition).

H. pylori exhibits some genetic diversity in the CagA sequence, so it may be useful to use a peptide or group of peptides capable of identifying multiple strains. SEQ ID NO 1 to SEQ ID NO 7 represent such a group of peptides, since 95% of all CagA+ H. pylori isolates worldwide have at least one of these sequence variants. Therefore, it may be useful to provide a mixture (a "cocktail") of two or more peptides from this document (SEQ ID NO 1-330). In one embodiment, said mixture comprises at least two, preferably three, more preferably four, more preferably five, more preferably six, and most preferably seven, peptides selected from peptides comprising or consisting of SEQ ID NO 1 to SEQ ID NO 13. In one embodiment, the sequences are selected from SEQ ID NO 1 to SEQ ID NO 7. Preferred mixtures include SEQ ID NOs 1, 2, 3, 4, 5, 6 and 7, SEQ ID NOs SEQ ID NOs 1, 2, 3, 4 and 5, SEQ ID NOs 2, 3, 4, 5, 6 and 7 and SEQ ID NOs 2, 3, 4 and 5. SEQ ID NO 1 to SEQ ID NO 5 are present in the types designated CagA ABC, ABCC and ABOCO, while SEQ ID NO 6 and SEQ ID NO 7 are only present in the ABD type. Thus, in one embodiment, one sequence is selected from SEQ ID NOs 1 to 5 and one sequence is selected from SEQ ID NOs 6 and 7. The peptides of SEQ ID NOs 6 and 7 may be particularly useful for the diagnosis of H. pylori strains from Asia.

In another embodiment, the peptides are selected from SEQ ID NO 8 to SEQ ID NO 13.

Another useful way to detect more than one strain of H. pylori is to use a peptide containing the EPIYA motif, present in SEQ ID NO 8, 9, 10 and 12.

One or more peptides may be included in a kit. The kit may be used for diagnosis as described herein. A kit may comprise one or more peptides or mixtures thereof, a binding buffer, and detection agents such as a secondary antibody. The kit may include a substrate that binds the peptide, such as a solid support, microtiter plates or ELISA plates pre-impregnated with the peptide(s) of the present invention, various diluents and buffers, labeled conjugates or other agents for detecting specific binding of antigens or antibodies, such as secondary antibodies, and other signal-generating reagents, such as enzyme substrates, cofactors, and chromogens. Other suitable components of the kit will be readily determined by one of skill in the art.

EXAMPLES

Example 1

Relevant CagA peptides were identified by a three-step procedure using peptide array experiments. Antibody binding characteristics of the peptides were analyzed by incubating the arrays with pooled or individual serum samples from H. pylori-infected and -uninfected individuals from a cohort of patients with dyspepsia. H. pylori-infected individuals had infections with a known CagA status (presence/absence of the cagA gene).

Serum samples were obtained from individuals in Managua, Nicaragua, undergoing endoscopy for dyspepsia as previously described (Thorelle et al., BMC Evol Biol 2016;16:53). All of these patients had a known H. pylori infection status, and the genomic sequences of their H. pylori isolates were available.

The published genome sequences of H. pylori were obtained from NCBI. The available complete genomes (n = 49) of H. pylori were downloaded from GenBank in August 2013. The experimental strains B8, Rif1, Rif2, UM298, and UM299 were removed, and the remaining 44 complete strains were used for comparative genomics. The whole-genome sequenced isolates available from GenBank as of November 1, 2013, were downloaded, and all isolates containing open reading frame information were used, except for strains passaged in animals or experimentally derived. Previously published Nicaraguan genome sequences in the Sequence Read Archive database (http://www.ncbi.nlm.nih.gov/sra) were also used, under accession number SRP045449.

In addition to these publicly available genomic sequences, sequences from H. pylori strains isolated in Australia were obtained from Professor Barry J. Marshall (University of Western Australia, WA, Australia).

To identify deduced CagA protein sequences within the available genomes, a blastp similarity search was performed using the CagA sequence from strain 26695 (NC_000915.1). In our genome sequence collection, 245 strains/isolates were found to contain the cagA gene, and all deduced CagA protein sequences from these isolates were used for further analysis.

Example 2

Peptide array analysis was used to determine the antibody response against CagA peptides. Medium-density arrays were printed using laser printing synthesis technology. Approximately 8,600 different H. pylori peptides (15-mers) of 15 amino acids were deposited on each chip. The chips were subsequently incubated with a 1/1000 dilution of patient serum or with a 1/1000 dilution of a pool of 10 different serum samples, followed by washing and subsequent incubation with fluorochrome-conjugated rabbit anti-human IgG antibodies. Finally, fluorescence image scanning and digital image analysis were performed to detect antibody binding to each of the peptides on the chip. Chip printing and antibody analysis were performed by PEPperPRINT (Heidelberg, Germany).

Example 3

High-density arrays were created by on-chip photolithographic synthesis. In these experiments, approximately 200,000 different 15-mer H. pylori peptides were deposited on each chip. The chips were then incubated with a 1/1000 dilution of patient serum or a 1/1000 dilution of a pool of 10 different serum samples. They were then washed and incubated with fluorochrome-conjugated rabbit anti-human IgG or anti-human IgA antibodies. Finally, fluorescence image scanning and digital image analysis were performed to detect antibody binding to each of the peptides on the chip. Chip printing and antibody analysis were performed by Schafer-n (Copenhagen, Denmark).

Example 4 - Identification of CagA epitopes in B cells

Serum antibody binding to a set of overlapping 15-mer peptides and serum samples was assessed to screen the complete CagA sequence. Medium-density arrays from Example 2 were used with peptides covering the entire CagA sequence with a 10-amino acid sequence overlap (n = 234 peptides). In subsequent experiments, high-density arrays from Example 3 were used with 15-mer peptides covering the entire CagA sequence, but this time with a 14-amino acid sequence overlap (n = 1172 peptides). In both cases, H. pylori strain 26695 was used as the source of the CagA peptide sequences. Antibody binding to each peptide was assessed individually on the array, and two groups of sera were used: one composed of pooled sera from 10 H. pylori-infected individuals and the other composed of pooled sera from 10 H. pylori-infected individuals. pylori (Hp+) and the other composed of sera from 10 uninfected individuals (Hp-).

Antibody binding of the Hp+ sera group was compared with that of the Hp- group. Linear B-cell epitopes were defined as stretches of at least four amino acids where antibody binding in the Hp+ group was at least twice as high as in the Hp- group. In this way, it was determined that H. pylori CagA contained 18 different linear B-cell epitopes, with an average length of 22 amino acids (Table 1 and Fig. 1). All of these epitopes are useful for the diagnosis of H. pylori CagA+ infection.

TABLE 1

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Example 5 - Identification of 15-mer CaqA peptides with high diagnostic potential

Individual serum samples were analyzed for antibody binding to the identified epitopes in order to assess the frequency of H. pylori -infected individuals with or without CagA and the antibody response to the different epitopes. Because the 18 epitopes each spanned more than one 15-mer peptide, overlapping peptides were again used, this time with a 10- or 11-amino acid overlap between the peptides in the sequence. In addition, given the considerable diversity of CagA sequences in different H. pylori isolates, sequence variants of each peptide were included. Therefore, for each overlapping 15-mer peptide sequence derived from 26,695 CagAs, all available sequence variants of those peptides were also used, if those sequence variants were determined to be present at least twice in our database of 245 CagA sequences from around the world. In total, 1,144 different CagA peptides and sequence variants within the 18 epitopes identified were analyzed using high-density arrays. All peptides were analyzed with individual serum samples (n = 48) from individuals with or without CagA+ H. pylori infection, as well as from an uninfected control group.

Epitopes with a high response frequency and strong antibody binding in individuals are suitable for diagnosing CagA+ H. pylori infection. One problem with previously known methods for assessing CagA antibodies was the high number of false positives (i.e., individuals not infected with H. pylori) who tested positive. Therefore, peptides with good discriminatory ability were identified: a strong antibody response in CagA+ infected individuals, but a minimal response in those infected without CagA and in individuals not infected with H. pylori.

The discriminatory capacity of the peptides was analyzed by ROC curves, using the area under the curve (AUC) of the ROC curve as an estimate of the diagnostic capacity.

The median AUC-ROC of 1144 different peptides from the 18 identified CagA epitopes, including sequence variants, was 0.53 (Fig. 2A). Since an AUC-ROC of 0.53 is very similar to the diagnostic accuracy of a coin toss (i.e., not diagnostically useful), this highlights the problem of the high false-positive rate in existing serological tests that rely on antibody responses against the full-length CagA protein.

An uneven distribution of ROC AUC was observed among the different epitopes, with epitopes 3-4, 8-14 and 17-18 containing the majority of peptides with high diagnostic capacity (Fig. 2B).

Of the 1144 peptides, 176 CagA peptides were identified with an ROC AUC greater than 0.7 (Table 2). Each of these peptides can be used for the diagnosis of CagA+ H. pylori infection.

TABLE 2

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Example 6

Even a diagnosis with an ROC AUC of less than 0.7 can have diagnostic value. To assess this, peptides were identified to which a consistent proportion of CagA+ individuals mounted an antibody response, but to which none of the individuals with CagA-negative strains or without H. pylori infection mounted such a response. Thus, 123 peptides were identified with an ROC AUC of less than 0.7, with a true-positive rate greater than 10% and a false-positive rate of 0% (Table 3). Each of these peptides can also be used for the diagnosis of CagA+ H. pylori infection.

TABLE 3

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Example 7 - Identification of amino acid sequences crucial for the diagnosis of H. pylori CaaA+ infection.

Detailed mapping of H. pylor CagA B cell epitopes was performed on peptides identified as highly diagnostic. Mapping was performed using high-density peptide arrays. Individual serum samples (n = 48) were analyzed for antibody binding to sequence variants of each of the selected peptides. This was done to identify amino acid positions within each peptide that contributed to antibody binding and were therefore critical for diagnostic applications.

The peptides with the greatest diagnostic potential were selected, and for each of them, 300 different sequence variants were created. This was carried out using what is known as a complete single-residue substitution. This means that, for each of the 15 amino acid positions in each peptide, the inventors created 20 different sequence variants that differed only in the sequence at that position; at that position, the 20 variants had one of each of the 20 common amino acids in the proteins. Since there were 20 different sequence variants for each amino acid position and the peptides were 15 amino acids long, a total of 300 different sequence variants were obtained. This procedure has been described previously (Hanse et al., PLOS One 2013:8(7):e68902). This analysis determined whether the position of a residue within the peptide is irrelevant to the binding of the peptide to the antibody, that is, whether amino acid residues in the native sequence can be freely substituted without affecting binding.

In this way, antibody binding of all variants of the selected peptides in each of the 48 serum samples was analyzed. The inventors observed that some peptide variants obtained AUC-ROC scores significantly/substantially lower than those of the original peptide and, based on this information, were able to identify sequence motifs crucial for the discriminatory ability of H. pylori CagA+ infection.

This revealed that the crucial portions of the peptides span 5–6 amino acids, and that redundancy exists at some positions within these crucial sequences. The crucial sequences of some epitopes are shown in Table 4, and their AUC-ROC levels are shown in Fig. 3. The peptides in Table 4 are particularly useful for the diagnosis and treatment of H. pylori infection and gastric cancer, including cancer prevention, because they are highly specific.

TABLE 4

Claims (7)

1. A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO 1 to SEQ ID NO 5, said peptide consisting of a maximum of 25 amino acids, for use in an in vivo method for diagnosing Helicobacter pylori infection and predicting the risk of gastric cancer. 2. The peptide for use according to claim 1, wherein the peptide is selected from the group consisting of SEQ ID NO 2 to SEQ ID NO 5. 3. The peptide for use according to any one of claims 1 to 2, wherein the peptide comprises a maximum of ten amino acids. 4. The peptide for use according to any one of claims 1 to 4, wherein the peptide consists of an amino acid sequence selected from SEQ ID NO 1 to SEQ ID NO 5. 5. A diagnostic method comprising the steps of: a) provide a sample obtained from a subject, the subject being a human being; b) contacting said sample with a peptide comprising an amino acid sequence selected from SEQ ID NO 1 to SEQ ID NO 5, said peptide consisting of a maximum of 25 amino acids; c) detecting the specific binding of the antibodies in the sample to the peptide, wherein the method is not practiced in a human body, wherein the method is used for the detection of Helicobacter pylori infection or the prediction of gastric cancer risk. 6. A method for detecting CagA-binding antibodies of H. pylori in a sample obtained from a subject, the method comprising contacting a biological sample with a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO 1 to SEQ ID NO 5, said peptide consisting of a maximum of 25 amino acids, and detecting the binding of the antibodies in the sample to the peptide. 7. The method according to claim 6, wherein the sample is a blood, serum, plasma or gastric tissue sample.